Nicotine-Mediated Recruitment of GABAergic Neurons to a Dopaminergic Phenotype Attenuates Motor Deficits in an Alpha-Synuclein Parkinson’s Model

Previous work revealed an inverse correlation between tobacco smoking and Parkinson’s disease (PD) that is associated with nicotine-induced neuroprotection of dopaminergic (DA) neurons against nigrostriatal damage in PD primates and rodent models. Nicotine, a neuroactive component of tobacco, can directly alter the activity of midbrain DA neurons and induce non-DA neurons in the substantia nigra (SN) to acquire a DA phenotype. Here, we investigated the recruitment mechanism of nigrostriatal GABAergic neurons to express DA phenotypes, such as transcription factor Nurr1 and DA-synthesizing enzyme tyrosine hydroxylase (TH), and the concomitant effects on motor function. Wild-type and α-syn-overexpressing (PD) mice treated with chronic nicotine were assessed by behavioral pattern monitor (BPM) and immunohistochemistry/in situ hybridization to measure behavior and the translational/transcriptional regulation of neurotransmitter phenotype following selective Nurr1 overexpression or DREADD-mediated chemogenetic activation. We found that nicotine treatment led to a transcriptional TH and translational Nurr1 upregulation within a pool of SN GABAergic neurons in wild-type animals. In PD mice, nicotine increased Nurr1 expression, reduced the number of α-syn-expressing neurons, and simultaneously rescued motor deficits. Hyperactivation of GABA neurons alone was sufficient to elicit de novo translational upregulation of Nurr1. Retrograde labeling revealed that a fraction of these GABAergic neurons projects to the dorsal striatum. Finally, concomitant depolarization and Nurr1 overexpression within GABA neurons were sufficient to mimic nicotine-mediated dopamine plasticity. Revealing the mechanism of nicotine-induced DA plasticity protecting SN neurons against nigrostriatal damage could contribute to developing new strategies for neurotransmitter replacement in PD.


Significance Statement
Tobacco smoking and Parkinson's disease (PD) have been associated with a mechanism of nicotine-induced neuroprotection of dopaminergic (DA) neurons against nigrostriatal (SN) damage. This study revealed that nicotine exposure led to a transcriptional TH and translational Nurr1 upregulation within a pool of SN GABAergic neurons in wildtype animals. In PD mice, nicotine treatment increased Nurr1 expression, reduced the number of α-syn-expressing neurons, and simultaneously rescued motor deficits. We successfully induced DA expression within SN GABA neurons in the absence of nicotine exposure by prolonged depolarization and concomitant Nurr1 overexpression. Revealing the recruitment mechanism of nigrostriatal GABAergic neurons to express DA phenotypes
Nicotine activates nicotinic acetylcholine receptors (nAChRs) and regulates the function of neurons by increasing calcium influx and inducing neuronal depolarization [21,22]. Nicotine-mediated calcium signaling occurs via direct calcium influx through nAChRs, indirect calcium influx through voltage-dependent calcium channels, and intracellular calcium release from internal stores [23][24][25]. A number of nAChR subtypes are expressed in both DAergic and GABAergic neurons in the SNc and SNr [26][27][28] and nicotine-induced neuroprotection can be mediated by heteromeric α4* (primarily α4β2*) and homomeric α7 receptors [29,30]. Importantly, chronic nicotine exposure upregulates α4* nAChRs localized in SNr GABAergic neurons without changing the α4* nAChRs levels in SNc DAergic neurons [28], suggesting that nicotine might initiate selective activity-dependent signaling on SNc and SNr neurons during chronic exposure.
The expression of the transcription factor Nurr1 (NR4A2), which is essential for the acquisition [31] and maintenance [32] of the DAergic phenotype, might participate in the mechanism of nicotine-mediated neuroprotection of nigrostriatal neurons. Studies have shown that Nurr1 expression is regulated by calcium-mediated neuronal activity [33] and increases in the striatum in response to chronic nicotine administration [34]. Importantly, Nurr1 plays a significant role in neuronal survival [35] and NR4A-deficient neurons are generally more sensitive to neurodegeneration due to the downregulation of NR4A-dependent neuroprotective gene programs [36]. Emerging evidence indicates that impaired Nurr1 expression might contribute to the pathogenesis of PD [37,38]. Due to its neuroprotective role for DAergic neurons, Nurr1 has been identified as a therapeutic target for PD. Remarkably, it was found that Nurr1 agonists improve behavioral deficits in a PD rat model [39]. Preclinical studies have also shown a promising role of Nurr1 in next-generation PD treatments, including Nurr1-activating compounds and Nurr1 gene therapy aimed at enhancing DA neurotransmission and protecting DAergic neurons from cell damage by environmental toxins and neuroinflammation [37,40,41].
Here, we investigate the cell-specific mechanism through which chronic nicotine influences the activity-dependent regulation of genes controlling the expression of Nurr1 and the DA-synthesizing enzyme tyrosine hydroxylase (TH) in neurons of the SN, while attenuating some of the PD-associated locomotor deficits. We then tested whether artificially recreating the cellular environment that mimics nicotine-mediated exposure in untreated wild-type mice is sufficient to induce dopamine plasticity in the SN.

Nicotine-Induced Neurotransmitter Plasticity Occurs via Translational Induction of Nurr1 and Transcriptional TH Regulation in Non-DAergic Neurons
To understand the regulatory mechanism through which DAergic phenotype is acquired by non-DAergic SNr neurons in response to chronic nicotine exposure, we first evaluated the protein expression of TH, the glutamate-decarboxylase-67 (GAD67) labeling GABAergic cells, and the transcription factor Nurr1 across conditions ( Figure 4A,B). Nurr1 protein, which is essential for the acquisition and maintenance of the DAergic phenotype [31,32], was detected in SNc TH+ neurons as expected. However, it was also clearly To determine whether the nicotine-mediated increase in the number of TH-expressing neurons occurs through recruitment of pre-existing SNr neurons to such a DAergic phenotype, we tested the effects of 2-week nicotine exposure on the SN of adult wildtype mice (P60). After nicotine exposure, brain tissue was labelled with the DAergic TH, neuronal NeuN, and nuclear DRAQ5 IHC markers. As observed in Pitx3-IRES2-tTA/tetO-A53T transgenic mice ( Figure 2E), stereological quantification indicated that chronic nicotine exposure in wild-type mice significantly increased the number of TH+ cells ( Figure 3A, arrows, inset) in the SNr (mean ± SEM: control = 101 ± 5, nicotine = 148 ± 7, t (19) = 5.01, p < 0.0001, Figure 2C), but not in the SNc ( Figure 3B). All SNr TH-expressing cells in nicotine-treated mice expressed Nurr1 ( Figure 3D, inset) and NeuN ( Figure 3E, inset) markers. The increased number of TH+ neurons was not due to an increase of neuroproliferation or cell migration, as no change in the total number of DRAQ5+ (mean ± SEM: control = 46 ± 5, nicotine = 42 ± 5) and NeuN+ cells (mean ± SEM: control = 16 ± 1, nicotine = 17 ± 1) was observed in the nicotine-exposed group ( Figure 3F,G).
We then performed RNAscope in situ hybridization (ISH; Figure 4D) to investigate whether the increased level of Nurr1 and TH protein expression took place via transcriptional or translational regulation. We found no difference in the total number of Nurr1-ISH+ cells across groups ( Figure 4D arrowheads and Figure 4E, mean ± SEM: control = 29 ± 3, nicotine = 33 ± 4) indicating that the increased number of Nurr1+ neurons observed in response to chronic nicotine exposure resulted from translational upregulation. In contrast, the increased number of TH-ISH+ cells in nicotine-exposed mice ( Figure 4F, mean ± SEM: control = 4.25 ± 0.85, nicotine = 9.33 ± 0.88, t (5) = 4.06, p < 0.01) reveals de novo transcription of TH mRNA in pre-existing SNr non-DAergic neurons.

The Pool of Neurons Recruitable for Nicotine-Induced TH Plasticity Is GABAergic
Because the SNr is primarily composed of GABAergic cells [46], we utilized the vesicular GABA transporter (VGAT)-ZsGreen transgenic mice, which constitutively expresses the ZsGreen-fluorescent protein in all GABAergic cells, to determine the fraction of neurons expressing TH and Nurr1 in the control condition. Quantification of TH/VGAT colocalization revealed a coexpression of 27 ± 4% (mean ± SEM) in the SNc and 47 ± 4% (mean ± SEM) in the SNr ( Figure 5A, inset arrows; Figure 5B) in control conditions. To identify SNr neurons that are recruitable by nicotine exposure to a TH phenotype, we investigated the pool of VGAT-expressing neurons that co-localized with Nurr1 and NeuN ( Figure 5C). We found that the fraction of Nurr1+/VGAT+/NeuN+ neurons ( Figure 5C, arrowheads) represents 39 ± 2% (mean ± SEM) of all SNr VGAT+ neurons ( Figure 5D). This pool of SNr GABAergic neurons, which display the molecular marker Nurr1 even before nicotine exposure, could represent a readily available reserve pool for nicotine-induced TH acquisition and for targeted activity-dependent manipulations.

A Fraction of SNr GABAergic Nurr1+ Neurons Project to the Striatum
The nigrostriatal pathway, which is affected by neurodegeneration in PD, comprises DAergic neurons originating from the SNc and projecting to neurons located in striatum subnuclei. However, an additional fraction of nigrostriatal projections originates from GABAergic neurons located in the SNr [47][48][49]. To confirm the connectivity of GABAergic SNr-to-striatum projecting neurons, fluorescent RetroBeads (555 nm) were injected into the dorsal striatum ( Figure 6A) for a retrograde tracing of striatal neuronal terminals to their SN somata. RetroBead-labelled cell bodies localized in the SNr identified SNr-to-striatum projecting neurons ( Figure 6B). RetroBead accumulation was detected in both VGAT+/TH-( Figure 5B, arrows) and TH+ SNr somata ( Figure 6B inset, arrowhead), in addition to all TH+ SNc-to-striatum projecting neurons. The fraction of Nurr1+ GABAergic neurons projecting to the striatum could serve as a reserve neuronal pool with the potential to acquire the TH phenotype and in turn replenish DA function in PD.   New IHC evidence showed that DAergic neurons in the SNc display a rich dendritic arborization extending into the SNr [44]. Such anatomical connectivity is in agreement with previous studies demonstrating that SNr GABAergic neurons can be electrically excited by direct activation of D 1 and D 5 receptors mediated by DA release from SNc DAergic dendrites [50] and nicotine-mediated activation of nAChRs [26,28].

Selective Nurr1 Upregulation in GABAergic Cells Is Not Sufficient to Induce a TH Phenotype
Because the fraction of SNr GABAergic neurons projecting to the striatum represents a reserve pool that can acquire Nurr1 and TH phenotypes in response to nicotine-mediated activation, we tested whether Nurr1 upregulation alone, exclusively targeted to SN GABAergic cells, could induce TH plasticity. We unilaterally injected a Cre-dependent Nurr1 viral vector (AAV.FLEX.Nurr1) into the SN of VGAT-Cre mice (P60). The contralateral side was used as a control. Nurr1 immunoreactivity in the AAV.FLEX.Nurr1-injected side, as compared to the control, showed robust vector transduction ( Figure 8A, arrowheads). A quantitative analysis showed that the increase in the number of Nurr1-expressing VGAT+ cells ( Figure 8B, mean ± SEM: control = 54 ± 3, AAV.FLEX.Nurr1 = 98 ± 12, t (7) = 2.96, p < 0.05) was not paralleled by an increase in the number of TH+ neurons in the SNr ( Figure 8C, mean ± SEM: control = 11 ± 1, AAV.FLEX.Nurr1 = 12 ± 1), indicating that Nurr1 upregulation alone was not sufficient to induce the acquisition of TH phenotype by SNr GABAergic neurons.

Discussion
Our findings show that chronic nicotine exposure attenuates locomotor deficits in a human-α-syn-expressing mouse model of PD [42] and primes a GABAergic neuronal pool in the SNr to a novel form of neuroplasticity, culminating in the acquisition of the TH phenotype. Nicotine activation of nAChRs in the nigrostriatal pathway elicits an increase in calcium influx [23][24][25] and induces neuronal depolarization [21,22]. Previous studies have reported on the dense distribution of nAChRs in both DAergic and GABAergic neurons in the SN [26,28], suggesting a potential activity-dependent mechanism in the regulation of DAergic circuits in nicotine-mediated protection against PD [19,[56][57][58]. Specifically, 99% of SNr GABAergic neurons express both α 4 * nAChR readily available for nicotine activation [26,28] as well as DA D 1 and D 5 receptors which are tonically excited by dendritically released DA from the SNc DAergic neurons, forming a relatively short SNc-to-SNr DAergic pathway [50]. Such dendro-dendritic connectivity and the specific receptor expression displayed by descending SNc DAergic dendrites into the SNr GABAergic neuropil provide the opportunity for the nigrostriatal circuit to signal common instructions to both the DAergic and the GABAergic pathways when DA function needs a physiological boost. These conditions have been shown to be a requirement for activity-dependent recruitment of non-DAergic neurons to a DAergic phenotype [45]. Chronic nicotine exposure could elicit the recruitment of SNr GABAergic neurons to Nurr1 and TH phenotypes through at least two potential activity-dependent signaling mechanisms: (a) nicotine directly activates the α 4 * nAChRs localized on the SNr GABAergic neurons; or (b) as nicotine activates SNc DAergic neurons via nAChRs, the DA released from the dendrites activates SNr GABAergic neurons through D 1 and D 5 receptors. Both mechanisms could, in principle, initiate the calcium-mediated reprogramming required to induce the TH phenotype in the SNr GABAergic neurons, as previously found in neurons of the SNc [59].
Electrical activity and calcium signaling have significant roles in regulating various forms of neuroplasticity, including priming neurons with the molecular memory of early nicotine exposure [54] and neurotransmitter reprogramming [51,60,61]. Sustained alteration in circuit activation by either experimental manipulation or natural sensory stimuli can induce neurotransmitter plasticity in the brain, affecting behavior [52][53][54][62][63][64]. While SNr GABAergic neurons undergo a significant upregulation of α 4 * nAChR subtype in response to chronic nicotine exposure, the level of these receptors in SNc DAergic neurons remains unchanged [28]. Selective upregulation of α4* nAChR levels in the SNr might bring the level of calcium transients in these neurons to a threshold sufficient to signal and initiate neurotransmitter plasticity in response to chronic nicotine exposure, providing another layer of specificity in the recruitment of GABAergic neurons of the SNr and not the SNc to nicotine-mediated TH plasticity.
In this study, we identified a reserve pool of Nurr1-expressing GABAergic neurons in the SNr that undergoes nicotine-mediated TH respecification; a phenomenon that might represent a layer of functional protection against PD. Our findings provide an important parallel to previously reported phenotypic shift of pre-existing GABAergic neurons to express TH in adult macaques following treatment with MPTP, a neurotoxin that induces DA depletion mimicking PD [65]. Importantly, we confirmed by retrograde tracing that part of the nigrostriatal projection originates from SNr GABAergic neurons and demonstrated that these neurons share the same target as DAergic neurons in the SNc. Therefore, these SNr GABAergic neurons could serve the role as a reserve pool that could gain the DA-synthesizing enzyme and potentially rescue the DAergic loss of function caused by neurodegeneration of SNc DAergic neurons. Given that this form of TH plasticity also occurs in the SN of primates [66] in response to DAergic neuron loss, understanding the mechanism of nicotine-induced TH respecification in the SNr has tremendous translational value in the constant search for new approaches aimed at replenishing DA function in PD.
As chronic nicotine exposure leads to improved locomotion in hα-syn+ mice and concomitantly increases the number of Nurr1+ cells in the SNr, we further investigated the effect of the induced upregulation of Nurr1 expression in the SN of hα-syn+ mice. We found that Nurr1 overexpression was sufficient to ameliorate PD-related locomotor deficiencies. This is in agreement with previous studies highlighting the role of Nurr1 in pathogenesis of PD and its potential as a therapeutic target [37,38]. Our results here show, for the first time, that Nurr1 overexpression elicits protection against PD-related locomotor dysfunctions through a reduction of the number of hα-syn-expressing TH+ neurons. In addition, the upregulation of Nurr1/Foxa2 co-expression in non-DAergic neurons observed after chronic nicotine exposure could represent part of the priming mechanism generating a molecular memory of nicotine exposure aimed at expanding the reserve pool of potential neurons equipped to undergo the TH genetic program when properly motivated by a persistent nicotine exposure.
Given the established neurodegenerative effects of abnormal α-synuclein [67] and the therapeutic effect of Nurr1 [37,38], chronic nicotine exposure might slow down the etiology of neurodegeneration by reducing and attenuating α-syn toxicity.
Our chemogenetic approach implemented to selectively and chronically depolarize GABAergic neurons was not sufficient to induce TH plasticity in these neurons; however, it revealed a way to experimentally induce an expansion of the reserve pool of Nurr1+ neurons in the SNr that is available for recruitment to TH phenotype acquisition. Indeed, selective Nurr1 upregulation and chronic activation of GABAergic neurons together recapitulated TH respecification observed in the SNr of mice chronically exposed to nicotine.
Future studies will uncover all key players required for a combinatorial manipulation that would successfully induce SNr GABAergic neurons to acquire the TH phenotype even in PD mice. Since the SNr GABAergic fraction of the nigrostriatal pathway is completely spared by PD-associated neurodegeneration, the gain of the DAergic phenotype could in principle replenish DA in the striatum. Establishing effective manipulations targeted to induce TH plasticity in GABAergic neurons of the nigrostriatal pathway could represent a paradigm shift in developing a novel approach for PD treatment.

Mice
Mice were from a C57BL/6J genetic background, except for GAD67-GFP mice that were from a CD1 background. The Gad1-tm1.1Tama (GAD67-GFP knock-in) mouse line was provided by Y. Yanagawa (Gunma University Graduate School of Medicine, Japan). Mice were heterozygous for insertion of the gene encoding GFP under the control of the GAD67 gene promoter. They were used to label the inhibitory GABAergic neurons in the SN by enhancing the GFP signal with an anti-GFP antibody. Adult (P60) mice, obtained from the Jackson Laboratory (Bar Harbor, ME, USA) weighing 25-35 g, were used in this study. VGAT-IRES-Cre knock-in mice (STOCK Slc32a1tm2(cre)Lowl/J, Jackson stock 016962) were used for chemogenetic manipulations. To induce the expression of the ZsGreen label in GABAergic cell bodies, VGAT-Cre mice were bred with reporter mice (B6.Cg-Gt(ROSA)26Sor tm6(CAG−ZsGreen1)Hze /J, Jackson stock 007906) that express CAGpromoter-driven enhanced green fluorescent protein (ZsGreen1) following Cre-mediated recombination. The Pitx3-IRES2-tTA/tetO-A53T double transgenic mouse line, which expresses mutant (SNCA*A53T) human α-synuclein in midbrain dopaminergic neurons, was previously characterized [42] and generously provided by Dr. Cai at NIH. By crossing the driver line, Pitx3-IRES-tTA mice (B6.129(FVB)-Pitx3 tm1.1Cai /J, Jackson stock 021962), with the responder line, tetO-A53T, which encodes a human α-synuclein mutant gene under the control of a tetO promoter (STOCK Tg(tetO-SNCA*A53T)E2Cai/J, Jackson stock 012442), the expression of A53T α-synuclein in the SN dopaminergic neurons was driven using a binary tetracycline-dependent "tet-off" inducible gene expression system. Breeders were given doxycycline (DOX)-containing (200 mg/kg) food pellets (Bio-Serv, Flemington, NJ, USA), in place of a regular diet, to suppress transgene expression from early embryonic stages through weaning (P21). Adult mice, weighing 20-30 g, were used to investigate either the accumulation of human α-synuclein (P30 through P180) or the effects of nicotine exposure. Mice of the responder line tetO-A53T were used as controls (hα-syn−). Mice were housed in accordance with the guidelines of the University of California San Diego Institutional Animal Care and Use Committee. Experiments involved male and female adult mice (9 to 16 weeks old) maintained either in 12:12 light/dark cycles (12 h light and 12 h dark) with food and water available ad libitum.
For retrograde tracing fluorescent RetroBeads (80 nL, 555 nm, LumaFluor, Inc., Durham, NC, USA) were unilaterally injected in the striatum (AP = −0.20 mm, L = ±2.60 mm, DV = −3.00 mm) of VGAT-ZsGreen mice. Mice were sacrificed after 10 days of recovery to allow adequate time for retrograde transport of RetroBeads from the striatal terminals to the soma of SN neurons.

Drug Treatment and Blood Collection
For chronic nicotine exposure two groups of adult GAD67-GFP (P60), VGAT-Cre (P60), or hα-syn+ (P111) mice underwent chronic nicotine exposure for two weeks. Drinking water was replaced with a solution of 50 mg/L nicotine in 1% saccharin (nicotine-treated group) or with 1% saccharin solution (control condition). Animals were sacrificed after the two-week treatment. The amount of fluid intake was measured daily throughout the experiment; the initial and final body weight of the mice were also measured. Plasma For colorimetric DAB (3,3-Diaminobenzidine)-based IHC, free-floating sections were washed three times (10 min per wash) in PBS, then incubated in Avidin-Biotin Complex (Vector laboratories, Newark, CA, USA) solution (1X PBS containing 0.3% Triton X-100, 2% NaCl, and 1% of Reagents A and B from the vectastain ABC kit) for one hour, washed again (3 × 10 min), and incubated in fresh DAB solution (25 mg/mL) for approximately 3 min depending on the speed of the reaction. Sections were rinsed twice quickly and washed for 20 min in PBS before mounting on glass slides. Sections on slides were dried in a fume hood, then defatted in 1:1 chloroform:ethanol solution for two hours, and progressively rehydrated in 100% ethanol, 95% ethanol, and distilled water. Sections were then counterstained in 0.1% cresyl violet solution for 30 min, rinsed quickly in distilled water, dehydrated in 95% ethanol for three minutes, in 100% ethanol twice for five minutes each, and cleared (2 × 5 min) in Xylenes (brand). Slides were cover-slipped with permanent mounting medium Cytoseal TM 60 (Thermo Scientific, Waltham, MA, USA). Images were acquired using Hamamatsu Nanozoomer 2.0HT Slide Scanner. The quantification was performed by unbiased stereology (using a Leica DM4 B microscope and Stereologer 2000 software, version SS-15, MBF Bioscience, Williston, VT, USA).
An unbiased count of DAB-stained neurons was performed using a Leica DM4 B microscope and Stereologer2000 software. The investigator was blind to experimental conditions. An exhaustive count of SNc TH-immunostained neurons (Slab Sampling Interval = 1, Total Number of Sections = 20, Section Sampling Interval = 2) was performed with a 63X oil objective after outlining the SNc with a 10× objective. The count was performed using a total of 100 dissectors (Frame Area: 5000 µm 2 , Frame Height: 20 µm, Guard Height: 2 µm, Frame Spacing: 100 µm). A neuron was considered as positive for immunoreactivity when its nucleus fell inside the dissector borders without touching the exclusion lines. For SNr TH-immunoreactive neurons ( Figure 1E: Slab Sampling Interval = 1, Total Number of Sections = 24, Section Sampling Interval = 3; Figure 2C: Slab Sampling Interval = 1, Total Number of Sections = 20, Section Sampling Interval = 2), a rare event protocol was used to perform an exhaustive count with a 10× objective (Frame Area: 5000 µm 2 , Frame Height: 20 µm, Guard Height: 2 µm, Frame Spacing: 100 µm).

Behavioral Testing
The causal links between changes in DA expression and behavior have been documented for other DA networks [69]. To assess PD-related behavioral deficits associated with A53T-expression and effects of nicotine treatment, mouse behavioral pattern monitor (BPM, San Diego Instruments, San Diego, CA, USA) chambers were used to measure locomotor activity and investigatory behavior [7]. This system collects data encompassing total traveling distance, rearing movements, duration spent in the center, number of entries to the center, transitions (number of times mouse entered one of nine regions), and number of investigatory nosepokes (holepokes). A mouse BPM chamber is a clear Plexiglas box containing a 30 × 60 cm holeboard floor. Each chamber is enclosed in a ventilated outer box to protect it from outside ambient noise and light. The location of the mouse is obtained from a grid composed of a 12 × 24 X-Y array of infrared photobeams that are placed 1 cm above the floor. There are 8 square sectors (15.2 cm wide) in each chamber. Crossovers between each sector are defined as movements between any of these sectors. Each chamber is also divided into 9 regions unequal in size that are used primarily to define entries into the corners and the center. Rearing is detected by an array of 16 photobeams placed 2.5 cm above the floor. Holepokes are detected by 11 1.4 cm holes in the chamber (3 in the floor and 8 in the wall), each equipped with an infrared photobeam. The status of the photobeams is sampled every 55 ms. A change in the status triggers the storage of information in a binary data file together with the duration of the photobeam status. Subsequently, the raw data files are transformed into (x, y, t, event) ASCII data files composed of the (x, y) location of the animal in the mouse BPM chamber with a resolution of 1.25 cm, the duration of each event (t) and whether a holepoke or rearing occurred (event). ASCII data were then exported into Microsoft Excel files for subsequent statistical analyses with GraphPad Prism 8.4.0.
A total of eight chambers was used, each chamber measuring one mouse per session (40 min). The BPM test was conducted after 14 days of nicotine administration and performed over 2 days, with male mice tested on the first day and female mice on the second day to avoid disruption of behavior by scent from the opposite sex. The animals were brought into the testing room 1 h before testing. During testing, a white noise generator produced background noise at 65 dB. The chambers were cleaned thoroughly between testing sessions.

Experimental Design and Statistical Analysis
Data were analyzed using two-tailed Student's t-test and one-way, two-way, or mixed model analysis of variance (ANOVA), as appropriate for each experiment. A criterion based on z-score was used to detect outliers prior to running the ANOVA. The level 0.01 was chosen as the decision criterion for the z-score of 3.291 beyond which a datum is considered an outlier. Significant main effects and interactions were followed by Bonferroni's Multiple Comparisons tests. Data are represented by mean and standard error in bar and line graphs, or by the median and interquartile range with all data points in box and whisker plots. The alpha level was set to 0.05 for all analyses. Appropriate sample size for each experiment was determined with standard Cohens's d power analysis with target power set to 0.

Institutional Review Board Statement:
The UCSD Institutional Animal Care and Use Committee (IACUC) has approved our Animal Use Protocol n. S15013 on 09/01/2020.

Data Availability Statement:
Research data is stored in our laboratory storage unit.